The present invention relates to culture media which provide useful environments for cellular development. More particularly this invention relates to defined culture media supplement containing constituents produced from non-traditional sources that, when added to culture media, avoid the problems of prior culture media.
For years media supplements for culturing mammalian embryonic cells have been derived from animal fluids, and in particular blood serum. While serum based media supplements have been somewhat effective for culturing certain types of cells and tissues, these media supplements have been found to be undesirable. One of the main reasons that these serum based media supplements are unattractive candidates for culturing cells is because of the possibility that the resulting media will contaminated with impurities, toxins, and infective agents found in the fluid from which the media is derived. Additionally, because the animals from which the blood is collected are different and live in differing environments, the fluids produced by these animals have different components at differing concentrations. One important aspect of serum based medium that has been recognized is the requirement of macromolecules in the medium. In an attempt to mimic serum based products, researchers have attempted to add synthetic macromolecules, such as polyvinyl alcohol, to replace the macromolecules in the serum, such as albumin. However, because serum is largely undefined chemically, removing the serum from culture media and attempting to replace only the larger molecules has produced culture media which are less than ideal or ineffective for many purposes because the media are missing essential components.
Accordingly, there is a need for culture media supplements which are as effective as culture media supplements based on blood products while at the same eliminating potential sources of contamination. Additionally there is a need for standardized culture media.
The present invention describes a novel, physiologically based completely defined supplement to culture media for mammalian embryonic cells and gametes. This medium supplement may be used with in vitro fertilization media, embryo transfer media and embryo cryopreservation media for the mammalian preimplantation embryo, as well as a supplement to media for the development of embryonic stem cells, or any other similar media well known in the art. This supplement contains recombinant human albumin (rHA), fermented hyaluronan (HYN) and/or citrate, and combinations thereof. Addition of this supplement to the culture medium results in equivalent development compared to media supplemented with serum albumin purified from blood.
The supplement may comprise recombinant human albumin (rHA) at any appropriate concentrations for the media in which it is to be used. As further described herein, the use of rHA rather than naturally occurring human serum albumin (HSA) has numerous advantages.
Typically, when the supplement comprises rHA, the supplement comprises between about 0.1 mg/ml to about 20.0 mg/ml of rHA based on the total volume of the medium to which the supplement is added. In one embodiment, about 0.5 mg/ml to about 5.0 mg/ml of rHA is added based on the total volume of the medium.
The efficacy of the supplement can be enhanced by adding fermented hyaluronan (HYN) to the supplement. The addition of the fermented HYN to the media supplement demonstrates positive results.
The phrase xe2x80x9cincreases the viability of gametes or embryonic cellsxe2x80x9d as used herein is defined as including the increased development of the embryos to the blastocyst stage in the culture, the ability to hatch from the zona pellucida is increased in vitro, and/or an increase in the overall viability of the embryo in embryo cultures when embryos are cultured in a medium containing the supplement of the present invention as compared to being cultured in the same medium without the supplement.
Furthermore, the addition of fermented HYN to the appropriate medium significantly affects the ability of the blastocysts to survive freezing. The use of fermented HYN has several advantages over the use of HYN from a naturally occurring warm blooded vertebrate source such as purified from rooster comb or umbilical cord. By utilizing fermented HYN rather than HYN from a warm blooded vertebrate source, the ability to control the safety and stability of the HYN from different sources and batches is greatly increased.
When present, the amount of fermented HYN will generally be at concentrations between about 0.1 mg/ml to about 5.0 mg/ml based on the total volume of the medium. In one embodiment the fermented HYN will be added to the medium at concentrations between about 0.125 mg/ml to about 1.0 mg/ml based on the total volume of the medium.
The supplement can be further augmented by the addition of citrate. In one embodiment, citrate and rHA are both added to the medium supplement, as it has been surprisingly and unexpectedly discovered that the addition of citrate to a medium supplement containing rHA allows the rHA to closely duplicate the properties of HSA or bovine serum albumin (BSA). The addition of the citrate has a further enhancing effect on the development of the cultured cellular material. Any citrate used for media that is well known in the art may be used, including, but not limited to, choline citrate, calcium citrate, citric acid, sodium citrate, and combinations thereof. In one embodiment, sodium citrate is used. The citrate is generally added at concentrations between about 0.1 mM and about 5.0 mM, based on the total volume of the medium. In one embodiment, the citrate is added at concentrations between about 0.1 mM and about 1.0 mM, based on the total volume of the medium.
The medium supplement of the present invention comprises rHA, fermented HYN and/or citrate in any useful combination. In one embodiment, the medium supplement, and the medium to which the medium supplement is added, is free from non-recombinant macromolecules or macromolecules purified from an animal source. In another embodiment, the medium supplement, and the medium to which the medium supplement is added, is free of non-recombinant HSA and/or non-fermented HYN.
This invention is directed to the medium supplement described above, media containing the medium supplement, a method of making the medium supplement, kits containing the medium supplement, and a method of growing embryonic material employing the medium supplement described herein.
The present invention includes a method of growing cellular material, in one embodiment embryos, employing the medium supplement described herein such that they can be included in medium at the start of culture, or can be added in a fed-batch or in a continuous manner. Moreover, the components of the medium supplement may be added together, or separately, at different stages of the media production.
This supplement can be added to any appropriate mammalian cellular material culture media well known in the art, including but not limited to, embryo culture media, embryo transfer media and embryo cryopreservation media (to include both freezing and vitrification procedures) for embryos from any mammalian species, and stem cell media. Any media that can support embryo or cell development could be used, which includes, by way of example only, bicarbonate buffered medium, Hepes-buffered or MOPS buffered medium or phosphate buffered saline. Examples of media are G1.2/G2.2, KSOM/KSOMaa, M16, SOF/SOFaa, MTF, P1, Earle""s, Hams F-10, M2, Hepes-G1.2, PBS and/or Whitten""s. (Gardner and Lane, 1999; Embryo Culture Systems; Handbook of In Vitro Fertilization, CRC Press, Editors: Trounson A O and Gardner D K, 2nd edition, Boca Raton, pp 205-264.)
The production of rHA is well known in the art. In one embodiment, rHA is obtained from genetically modified yeast which produce a human albumin protein. One such methodology for the production of rHA from yeast is taught in U.S. Pat. No. 5,612,197.
Fermented hyaluronan (HYN) is obtained by any process well known in the art. One such process is the continuous bacterial fermentation of Streptococcus equi. Hyaluronan is a naturally occurring polymer of repeated disaccharide units of N-acetylglucosamine and D-glucuronic acid. It is widely distributed throughout the body. Typically, the molecular weight of the fermented HYN is 2.3xc3x97106 kD. The production of HYN from Streptococcus is well known in the art, and any well known process can be used, including those disclosed in Cifonelli J A, Dorfman A. The biosynthesis of hyaluronic acid by group A Streptococcus: The uridine nucleotides of groups A Streptococcus. J. Biological Chemistry 1957; 228: 547-557; Kjems E, Lebech K. Isolation of hyaluronic acid from cultures of streptococci in a chemically defined medium. Acta Path. Microbiol. Scand. 1976 (Sect. B); 84: 162-164; and Markovitz A, et al. The biosynthesis of hyaluronic acid by group A Streptococcus. J. Biological Chemistry 1959; 234(9): 2343-2350.
Other compounds may be added to the medium supplement of the present invention. These include growth factors, as mammalian embryos and cells typically have many receptors for growth factors and the addition of such growth factors may increase the growth rate of the cultured material. Such growth factors include, but are not limited to, Insulin, typically in amounts of 0.1-100 ng/ml; IGF II, typically in amounts of 0.1-100 ng/ml; EGF, typically in amounts of 0.1-100 ng/ml; LIF, typically in amounts of 5-1000 U/ml; PAF, typically in amounts of 0.1-500 xcexcM; and combinations thereof. All amounts are based on the total volume of the media to which the medium supplement is added.
Medium supplement can be prepared in 2 ways, either as a separate medium supplement that is added to the media after media preparation, or the ingredients of the medium supplement can be added directly to the culture media during media preparation.
By way of example only, the medium supplement may be prepared on its own as follows. Medium supplement rHA may be made into a stock solution by adding either water, saline or medium to make a concentrated stock solution of between 50-500 mg/ml, usually 250 mg/ml. Alternatively, the solution can be obtained as a 250 mg/ml stock solution. Fermented HYN is reconstituted in water, saline or medium, to make a concentrated stock solution of between 10-500 mg/ml, usually 500 mg/ml. This is achieved by adding the water, saline or medium to a flask and adding the desired amount of HYN to the solution. The HYN is then dissolved by rigorous shaking or mixing using a stir bar. For a 500 mg/ml solution, 500 mg of HYN can be added to 1 ml of solution. Citrate is prepared as a stock solution by adding either water, saline or medium to make a concentrated stock solution of between 5-500 mM, usually 500 mM. For a 500 mM stock solution, 0.9605 g of citric acid is added to 10 ml of solution. The rHA, fermented HYN and citrate stocks are added together to make a single supplement solution that is added to the final medium as a 100xc3x97times concentrated stock. For 10 ml of medium, 100 xcexcl of the supplement is added.
rHA can be added directly to the culture medium as either a powder or as a stock solution. The following embodiment is presented by way of example only. The stock solution may added as 100 xcexcl of 250 mg/ml stock to 9.9 mls of medium. Fermented HYN may be added directly to the culture medium as either a powder or as a stock solution. As a powder, 1.25 mg of HYN may be added to 10 ml of medium. Alternatively, a 125 xcexcl of a 1% stock solution may be added to 9.9 ml of medium. Citrate may be added directly to the culture medium as either a powder or as a stock solution. As a powder, 9.6 mg may be added to 100 mls of medium, or alternatively, 100 xcexcl of a 50 mM stock may be added to 9.9 ml of medium.
All patents and publications cited herein are hereby incorporated by reference.
All ranges recited herein include all combinations and subcombinations included within that range limits; therefore, a range from xe2x80x9cabout 0.1 mg/ml to about 20.0 mg/milxe2x80x9d would include ranges from about 0.125 mg/ml to about 11.5 mg/ml, about 1.0 mg/ml to about 15.0 mg/ml, etc.
The medium supplement of the present invention solves several problems that persist in the art of culturing mammalian cells, tissues, embryos and other related cellular material. One problem with current media is that the cultured mammalian cellular material, particularly embryos, may become contaminated by contaminants such as prions and/or endotoxins found within macromolecular blood products such as human albumin. An advantage of the supplement of the present invention is that it eliminates the potential contamination associated with the use of blood products in media for culturing embryo and other mammalian cellular materials.
Another problem with current media is the difficulty in standardizing such media when using blood products such as serum albumin or other naturally occurring materials. Furthermore, the present invention makes it easier to purify the final cultured product, when the naturally occurring variations and contaminants within the blood products in the media are eliminated.
The present invention eliminates the inherent variation involved when using a biological protein which is often contaminated with other molecules and which differs significantly between different preparations and also between batches within the same preparation. Therefore, the use of recombinant molecules such as rHA enables the formulation of physiological media to be prepared in a standardized fashion. These preparations are endotoxin free, free of prions and are more physiologically compatible than media which are currently used. Current media contain other synthetic macromolecules, such as polyvinyl alcohol or polyvinyl pyrrolidone, which are unable to perform essential physiological functions, such as bind growth factors, and therefore the use of these media result in inferior development of mammalian cellular material.